|Year : 1993 | Volume
| Issue : 4 | Page : 173-175
Acid phosphatase and lipid peroxidation in human cataractous lens epithelium
Abhay R Vasavada1, Prajitha Thampi2, Savita Yadav2, UM Rawal2
1 Raghudeep Eye Clinic and Ophthalmic Research Laboratory, Ahmedabad, India
2 Dept. of Zoology, School of Sciences, Gujarat University, Ahmedabad, India
Abhay R Vasavada
Raghudeep Eye Clinic, Opp. Gurukul, Drive-in Road, Ahmedabad 380 009
Source of Support: None, Conflict of Interest: None
The anterior lens epithelial cells undergo a variety of degenerative and proliferative changes during cataract formation. Acid phosphatase is primarily responsible for tissue regeneration and tissue repair. The lipid hydroperoxides that are obtained by lipid peroxidation of polysaturated or unsaturated fatty acids bring about deterioration of biological membranes at cellular and tissue levels. Acid phosphatase and lipid peroxidation activities were studied on the lens epithelial cells of nuclear cataract, posterior subcapsular cataract, mature cataract, and mixed cataract. Of these, mature cataractous lens epithelium showed maximum activity for acid phosphatase (516.83 moles of p-nitrophenol released/g lens epithelium) and maximum levels of lipid peroxidation (86.29 O.D./min/g lens epithelium). In contrast, mixed cataractous lens epithelium showed minimum activity of acid phosphatase (222.61 moles of p-nitrophenol released/g lens epithelium) and minimum levels of lipid peroxidation (54.23 O.D./min/g lens epithelium). From our study, we correlated the maximum activity of acid phosphatase in mature cataractous lens epithelium with the increased areas of superimposed cells associated with the formation of mature cataract. Likewise, the maximum levels of lipid peroxidation in mature cataractous lens epithelium was correlated with increased permeability of the plasma membrane. Conversely, the minimum levels of lipid peroxidation in mixed cataractous lens epithelium makes us presume that factors other than lipid peroxidation may also account for the formation of mixed type of cataract.
Keywords: Lens epithelium - Cataract - Acid phosphatase - Lipid peroxidation
|How to cite this article:|
Vasavada AR, Thampi P, Yadav S, Rawal U M. Acid phosphatase and lipid peroxidation in human cataractous lens epithelium. Indian J Ophthalmol 1993;41:173-5
A monolayer of epithelial cells covers the anterior surface of the fibers that comprise the bulk of the lens. The epithelium is divided into subpopulations on the basis of their proliferative kinetics. The cells of the central zone overlying the anterior pole of the lens do not normally undergo mitosis but can divide in response to various stimuli, such as injury and hormones. Most of the cell proliferation in the lens occurs in a band of epithelial cells above the equator known as the germinative zone. The progeny of these divisions migrate to the transitional zone to elongate and form fiber cells. The differentiation of fibers is characterized by a sequence of morphological changes occurring as the fibers mature. These changes include cell elongation, organelle loss, formation of specialized cell junctions, and fiber denucleation.
Histological studies have demonstrated varied abnormalities unique to the anterior lens epithelium of each type of cataract. Pathogenesis of cataract is closely related to changes in lens metabolism.  Opacification is accompanied by ultrastructural changes indicative of cellular degeneration. Acid phosphatase, an acid hydrolase, has been postulated to be of importance in the breakdown of extracellular material and tissue debris resulting from injury and pathological conditions. sub Lens epithelial cells are capable of synthesizing and secreting acid hydrolases.
Besides oxidation of functional and structural proteins, peroxidation of lenticular plasma membrane could be one of the major events in the pathogenesis of cataract. Lipid peroxidation reaction is initiated when a reactive oxidizing species interacts with, and abstracts an allylic hydrogen atom from a polysaturated fatty acid to form a fatty acyl radical which in turn attacks an oxygen molecule to form a lipid peroxy radical. This acts as an initiator attacking another molecule of polysaturated fatty acid to continue the chain propogation steps of peroxidation. 
In this study, the biochemical analysis with reference to lipid peroxidation and activity of acid phosphatase of the lens epithelium of different cataractous lenses have been investigated.
| Materials and methods|| |
Cataractous anterior lens capsule with epithelium were obtained from patients undergoing endocapsular intraocular lens implantation. The lens capsule with epithelium was removed microscopically with the help of forceps after insertion of intraocular lens. The type of cataract was classified by a detailed slit-lamp biomicroscopic examination prior to surgery as follows: (1) nuclear sclerosis (Type I); (2) posterior sub-capsular cataract (Type IV); (3) mature cataract (Type V); and (4) nuclear with posterior subcapsular .(mixed type) cataract (Type I + IV).
Acid phosphatase was estimated by the method of Linhardt et al.  Ten epithelia of each type of cataract was pooled to obtain a single sample. The lens epithelium was homogenized in distilled water. One millilitre of acid substrate buffer (0.05 M citrate buffer; 5 x 10 -3 M p-nitrophenyl phosphate, pH 4.8) was incubated at 37°C for 5 to 10 minutes. Then, 2 ml of lens epithelial homogenate was added. The contents were mixed and incubated for 30 minutes. Four millilitre of 0.1 N NaOH was then added to arrest the reaction. In the blank tube the homogenate was added after the addition of 0.1 N NaOH. The absorbance of the yellow colour so developed was read at 420 nm on the DU-40 Beckman spectrophotometer. The activity of acid phosphatase was estimated with respect to quantity of p-nitrophenol released.
Lipid peroxidation was estimated by the method of Bhuyan et al.  Ten epithelia of each type of cataract was pooled to obtain a single sample. The homogenate was prepared in 5 ml of 10 mM potassium phosphate buffer (pH 7.4), containing butylated hydroxy toluene (BHT) and homogenized adding 1 ml of 20% trichloroacetic acid. The homogenate was heated in a water bath at 70°C for 10 minutes, and centrifuged for 10 minutes at 25°C. A 1-ml aliquot of the proteinfree supernatant was mixed with 0.5 ml of 0.5% (w/ v) aqueous 2-thiobarbituric acid (TBA) in a test tube, topped with a glass ball and heated in a boiling water bath for 10 minutes. The test tube was then cooled to room temperature and the absorbance of the pinkcoloured trimethine condensation product was measured spectrophotometrically at 533 nm.
| Results|| |
The lens epithelium of mature cataract showed maximum activity of acid phosphatase (516.83 ± 2.0 moles of p-nitrophenol released/g lens epithelium) followed by the epithelium of mixed type of cataract (222.61 ± 1.0). In the epithelium of the nuclear and subcapsular type of cataract, the activity was 110.56 ± 2.0 and 115.56 ± 2.0, respectively [Table - 1].
Maximum levels of lipid peroxidation was observed in the epithelium of mature type of cataract (86.29 ± 1.0 O.D./min/g lens epithelium), followed by the posterior subcapsular (35.85 ± 0.38), and nuclear cataractous epithelium (25 ± 1.0), respectively.
| Discussion|| |
Acid hydrolases play an important role in the process of tissue remodelling and reorganization that accompanies ongoing proliferative and degenerative activities, and it is probable that they play a similar role in a variety of pathological conditions of the lens. Pathological conditions in the ocular lens, whether experimentally induced or occurring due to ageing, lead to an increase in acid phosphatase content and perhaps of other hydrolases as well. 
Morphological studies on the epithelium of mature cataract have revealed the coexistence of areas of low cell density and areas of superimposed cells.  The high activity of acid phosphatase in the epithelium of mature cataract type shows that this enzyme is activated with the complete cellular disruption and successive enhancement of the process of tissue reorganization. This supports the postulation that in addition to the possible role of acid hydrolytic enzyme in tissue debris degradation, it is conceivable that the enzyme may affect other processes associated with wound healing such as cellular migration and initiation of DNA synthesis. 
During lipid peroxidation the reactive oxidizing species reacts with, and abstracts an allylic hydrogen atom from a polysaturated fatty acid to form a fatty acyl radical which in turn attacks an oxygen molecule forming a lipid peroxy radical. Increased permeability of the plasma membrane during cataract formation could therefore be correlated to the peroxidation of membrane lipids.
The formation of hydrophilic lipid hydroperoxides in the hydrophobic barrier of the lipid bilayers of plasma membrane alters permeability in cataract.10
Compared to the other cataractous types studied, mixed type of cataractous epithelium showed remarkably low levels of lipid peroxidation. We presume that factors other than lipid peroxidation may also account for the formation of mixed type of cataract.
Of all the types of cataractous epithelium studied, the highest levels of acid phosphatase and lipid peroxidation were observed in the mature type of cataract. This result is in concordance with the earlier reported histopathology of mature cataractous epithelium.  Earlier studies on the histopathology of the lens epithelium of different cataractous types have revealed necrotic changes characteristic to each cataract type. In mature cataractous lens epithelium incidence of areas of superimposed cells were observed.  In the present study, the highest activity of acid phosphatase was observed in mature cataractous epithelium. As acid phosphatase enzyme is activated with complete cellular disruption and successive process of tissue reorganization and wound healing, we have correlated the increased areas of superimposed cells with the high activity of acid phosphatase. It would therefore be of interest to correlate the increased levels of acid phosphatase and lipid peroxidation with increased areas of superimposed cells neighbouring the monolayered areas of epithelial cells showing cytoplasmic vacuolization, as detected in mature cataractous epithelium.
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[Table - 1]